A mechanism is proposed in which the histidines residues His 354 and His 358 catalyze the formation of the four-membered ring intermediate in the repair process of this enzyme. When deuterium oxide is used as a solvent, the repair activity is decreased. The proton transfer shown by this isotope effect supports the proposed mechanism

The overall repair reaction consists of two distinct steps, one of which is light-independent and the other one light-dependent. In the initial light-independent step, a 6-iminium ion is thought to be generated via proton transfer induced by two histidines highly conserved among the (6-4) photolyases.This intermediate spontaneously rearranges to form an oxetane intermediate by intramolecular nucleophilic attack. In the subsequent light-driven reaction, one electron is believed to be transferred from the fully reduced FAD cofactor (FADH-) to the oxetane intermediate thus forming a neutral FADH radical and an anionic oxetane radical, which spontaneously fractures. The excess electron is then back-transferred to the flavin radical restoring the fully reduced flavin cofactor and a pair of pyrimidine bases

The overall repair reaction consists of two distinct steps, one of which is light-independent and the other one light-dependent. In the initial light-independent step, a 6-iminium ion is thought to be generated via proton transfer induced by two histidines highly conserved among the (6-4) photolyases.This intermediate spontaneously rearranges to form an oxetane intermediate by intramolecular nucleophilic attack. In the subsequent light-driven reaction, one electron is believed to be transferred from the fully reduced FAD cofactor (FADH-) to the oxetane intermediate thus forming a neutral FADH radical and an anionic oxetane radical, which spontaneously fractures. The excess electron is then back-transferred to the flavin radical restoring the fully reduced flavin cofactor and a pair of pyrimidine bases

The overall repair reaction consists of two distinct steps, one of which is light-independent and the other one light-dependent. In the initial light-independent step, a 6-iminium ion is thought to be generated via proton transfer induced by two histidines highly conserved among the (6-4) photolyases.This intermediate spontaneously rearranges to form an oxetane intermediate by intramolecular nucleophilic attack. In the subsequent light-driven reaction, one electron is believed to be transferred from the fully reduced FAD cofactor (FADH-) to the oxetane intermediate thus forming a neutral FADH radical and an anionic oxetane radical, which spontaneously fractures. The excess electron is then back-transferred to the flavin radical restoring the fully reduced flavin cofactor and a pair of pyrimidine bases

The overall repair reaction consists of two distinct steps, one of which is light-independent and the other one light-dependent. In the initial light-independent step, a 6-iminium ion is thought to be generated via proton transfer induced by two histidines highly conserved among the (6-4) photolyases.This intermediate spontaneously rearranges to form an oxetane intermediate by intramolecular nucleophilic attack. In the subsequent light-driven reaction, one electron is believed to be transferred from the fully reduced FAD cofactor (FADH-) to the oxetane intermediate thus forming a neutral FADH radical and an anionic oxetane radical, which spontaneously fractures. The excess electron is then back-transferred to the flavin radical restoring the fully reduced flavin cofactor and a pair of pyrimidine bases

The overall repair reaction consists of two distinct steps, one of which is light-independent and the other one light-dependent. In the initial light-independent step, a 6-iminium ion is thought to be generated via proton transfer induced by two histidines highly conserved among the (6-4) photolyases.This intermediate spontaneously rearranges to form an oxetane intermediate by intramolecular nucleophilic attack. In the subsequent light-driven reaction, one electron is believed to be transferred from the fully reduced FAD cofactor (FADH-) to the oxetane intermediate thus forming a neutral FADH radical and an anionic oxetane radical, which spontaneously fractures. The excess electron is then back-transferred to the flavin radical restoring the fully reduced flavin cofactor and a pair of pyrimidine bases

The overall repair reaction consists of two distinct steps, one of which is light-independent and the other one light-dependent. In the initial light-independent step, a 6-iminium ion is thought to be generated via proton transfer induced by two histidines highly conserved among the (6-4) photolyases.This intermediate spontaneously rearranges to form an oxetane intermediate by intramolecular nucleophilic attack. In the subsequent light-driven reaction, one electron is believed to be transferred from the fully reduced FAD cofactor (FADH-) to the oxetane intermediate thus forming a neutral FADH radical and an anionic oxetane radical, which spontaneously fractures. The excess electron is then back-transferred to the flavin radical restoring the fully reduced flavin cofactor and a pair of pyrimidine bases

using quantum mechanics/molecular mechanics studies, a repair mechanism is proposed, which involves two photoexcitations. The flavin chromophore, initially being in its reduced anionic form, is photoexcited and donates an electron to the (6-4) form of the photolesion. The photolesion is then protonated by the neighboring histidine residue and forms a radical intermediate. A second photoexcitation of the flavin promotes another electron transfer to the oxetane. Proton donation from the same histidine residue allows for the splitting of the four-membered ring, hence opening an efficient pathway to the final repaired form

time-resolved measurements are performed of radical formation, diffusion, and protein conformational changes during light-dependent repair by full-length (6-4) photolyase on DNA carrying a single UV-induced damage. The (6-4) photolyase shows significant volume changes after blue-light activation, indicating protein conformational changes distant from the flavin cofactor. A drastic diffusion change is observed only in the presence of both (6-4) photolyase and damaged DNA, and not for (6-4) photolyase alone or with undamaged DNA

ultrafast spectroscopy is used to show that the key step in the repair photocycle is a cyclic proton transfer between the enzyme and the substrate. By femtosecond synchronization of the enzymatic dynamics with the repair function, direct electron transfer from the excited flavin cofactor to the 6-4 photoproduct is observed in 225 ps but fast back electron transfer in 50 ps without repair. The catalytic proton transfer between a histidine residue in the active site and the 6-4 photoproduct, induced by the initial photoinduced electron transfer from the excited flavin cofactor to 6-4 photoproduct, occurs in 425 ps and leads to 6-4 photoproduct repair in tens of nanoseconds

repair photocycle of (6-4) thymine photoproduct by (6-4) photolyase, which involves light absorption by the 8-HDF cofactor and transfer of the excitation energy to the FADH-. Intermolecular Coulombic decay resulting in an electron transfer from FADH? to the dimer initiating the splitting process, mechanism, detailed overview. The mechanism requires the C5-OH transfer from C5 of the 5' thymine to the C4' of the 3' thymine followed by H transfer to the N3' of the 3' thymine. Of two conserved histidine residues, only His365 is protonated

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SYSTEMATIC NAME

IUBMB Comments

(6-4) photoproduct pyrimidine-lyase

A flavoprotein (FAD). The overall repair reaction consists of two distinct steps, one of which is light-independent and the other one light-dependent. In the initial light-independent step, a 6-iminium ion is thought to be generated via proton transfer induced by two histidines highly conserved among the (6-4) photolyases. This intermediate spontaneously rearranges to form an oxetane intermediate by intramolecular nucleophilic attack. In the subsequent light-driven reaction, one electron is believed to be transferred from the fully reduced FAD cofactor (FADH-) to the oxetane intermediate thus forming a neutral FADH radical and an anionic oxetane radical, which spontaneously fractures. The excess electron is then back-transferred to the flavin radical restoring the fully reduced flavin cofactor and a pair of pyrimidine bases [2].

the enzyme belongs to the photolyase/cryptochrome family, a large family of flavoproteins that possess different functions and use blue light as an energy source, phylogenetic analysis. Of the seven members of this gene family, three (CmPHR2, CmPHR5 and CmPHR6) fall within the clade of cryptochrome DASH, three (CmPHR3, CmPHR4 and CmPHR7) group with plant cryptochromes, and one (CmPHR1) is a homologue of (6-4) photolyase. Photolyases repair UV-induced DNA damage, whereas cryptochromes regulate the growth and development of plants in a blue-light dependent manner

(6-4)photolyases are broadly distributed in prokaryotes. the PhrB-like photolyases branched at the base of the evolution of the cryptochrome/photolyase family. The prokaryotic (6-4) photolyases are the ancestors of the cryptochrome/photolyase family

(6-4)photolyases are broadly distributed in prokaryotes. the PhrB-like photolyases branched at the base of the evolution of the cryptochrome/photolyase family. The prokaryotic (6-4) photolyases are the ancestors of the cryptochrome/photolyase family

the enzyme belongs to the photolyase/cryptochrome family, a large family of flavoproteins that possess different functions and use blue light as an energy source, phylogenetic analysis. Of the seven members of this gene family, three (CmPHR2, CmPHR5 and CmPHR6) fall within the clade of cryptochrome DASH, three (CmPHR3, CmPHR4 and CmPHR7) group with plant cryptochromes, and one (CmPHR1) is a homologue of (6-4) photolyase. Photolyases repair UV-induced DNA damage, whereas cryptochromes regulate the growth and development of plants in a blue-light dependent manner

the His365-His366-X-X-Arg369 motif is located within the proposed DNA lesion contact site of PhrB. This motif is structurally conserved in eukaryotic (6-4) photolyases for which the second His is essential for the (6-4) photolyase function. His366 in PhrB is stabilized by van der Waals contacts with Leu370 and Met410. The enzyme has a C-terminal extension

the His365-His366-X-X-Arg369 motif is located within the proposed DNA lesion contact site of PhrB. This motif is structurally conserved in eukaryotic (6-4) photolyases for which the second His is essential for the (6-4) photolyase function. His366 in PhrB is stabilized by van der Waals contacts with Leu370 and Met410. The enzyme has a C-terminal extension

the His365-His366-X-X-Arg369 motif is located within the proposed DNA lesion contact site of PhrB. This motif is structurally conserved in eukaryotic (6-4) photolyases for which the second His is essential for the (6-4) photolyase function. His366 in PhrB is stabilized by van der Waals contacts with Leu370 and Met410. The enzyme has a C-terminal extension

although the affinity of the enzyme for the Dewar photoproduct-containing duplex is similar to that for the (6-4) photoproduct containing substrate a repair rate could not be shown. These results indicate that the (6-4) photolyase binds the DNA containing the Dewar photoproduct and induces a structural change in DNA to some extent, suggesting a difference in the binding mode compared to the (6-4) photoproduct

A T(6-4)C photoproduct is synthesized. Differences from T(6-4)T is formation of cytosine hydrates by UV irradiation, and acylation of the amino function with the capping reagent. The capping step is omitted to improve the yield of the desired oligonucleotides. (6-4) photolyase restores the pyrimidines in T(6-4)C to their original structures

substrates are single stranded or double stranded DNA probe comprising the AGGT(6-4)TGGC or GCGGT(6-4)TGGCG paired with TCGCCAACCGCT. PhrB is active with ssDNA and dsDNA TT (6-4) photoproducts, substrate binding structure, detailed overview. Arg183 is part of the loop region connecting alpha7 and alpha8

substrates are single stranded or double stranded DNA probe comprising the AGGT(6-4)TGGC or GCGGT(6-4)TGGCG paired with TCGCCAACCGCT. PhrB is active with ssDNA and dsDNA TT (6-4) photoproducts, substrate binding structure, detailed overview. Arg183 is part of the loop region connecting alpha7 and alpha8

(6-4) photolyases repair (6-4) pyrimidine-pyrimidone photoproducts. CmPHR1 exhibits a repair activity of both (6-4)-ssDNA and (6-4)-dsDNA. Cryptochromes CmPHR2 and CmPHR5 show CPD repair activity only on ssDNA, and have no repair activity when CPD-damaged dsDNA is used as a substrate

(6-4) photolyases repair (6-4) pyrimidine-pyrimidone photoproducts. CmPHR1 exhibits a repair activity of both (6-4)-ssDNA and (6-4)-dsDNA. Cryptochromes CmPHR2 and CmPHR5 show CPD repair activity only on ssDNA, and have no repair activity when CPD-damaged dsDNA is used as a substrate

binding and catalytic properties of the enzyme are investigated using natural substrates, T[6-4]T and T[6-4]C, and the Dewar isomer of (6-4) photoproduct and substrate analogs s5T[6-4]T/thietane, mes5T[6-4]T, and the N-methyl-3’T thietane analog of the oxetane intermediate. The enzyme binds to the natural substrates and to mes5T[6-4]T with high affinity and produces a DNase I footprint of about 20 base pairs around the photolesion. Of the four substrates that bind with high affinity to the enzyme, T[6-4]T and T[6-4]C are repaired with relatively high quantum yields compared with the Dewar isomer and the mes5T[6-4]T which are repaired with 300-400-fold lower quantum yield

cyclobutane pyrimidine dimer-photolyase (EC 4.1.99.3) or 6-4PP-photolyase are able to prevent UV-induced apoptosis in cells deficient for nucleotide excision repair to a similar extent, while in nucleotide excision repair-proficient cells UV-induced apoptosis is prevented only by cyclobutane pyrimidine dimer-photolyase, with no effects observed when pyrimidine-(6-4)-pyrimidone photoproducts are removed by the specific photolyase. Both cyclobutane pyrimidine dimers and pyrimidine-(6-4)-pyrimidone photoproducts contribute to UV-induced apoptosis in nucleotide excision repair-deficient cells, while in nucleotide excision repair-proficient cells, cyclobutane pyrimidine dimers are the only lesions responsible for UV-killing, probably due to the rapid repair of pyrimidine-(6-4)-pyrimidone photoproducts by nucleotide excision repair

(6-4) photolyase is examined by optical spectroscopy, electron paramagnetic resonance, and pulsed electron nuclear double resonance spectroscopy. It is suggested that His354 and His358 catalyze the formation of the oxetane intermediate that precedes light-initiated DNA repair. At pH 9.5 where the enzyme repair activity is highest His358 is deprotonated, whereas His354 is protonated, acting as the proton donor that initiates oxetane formation from the (6-4) photoproduct

2-thio analog of the the (6-4) photoproduct, in which the carbonyl group at the C2 of the 3'pyrimidone is replaced with a thiocarbonyl group, is not repaired by the (6-4) photolyase. Cationic imine analogue of the (6-4) photoproduct, in which the carbonyl group at the C2 of the 3'pyrimidone is replaced with an imine (T(6-4)TNH2), is not repaired by the (6-4) photolyase, even in the presence of a 10 molar excess of the enzyme. 3'carbonyl group of the (6-4) photoproduct is involved in the recognition and reaction of the (6-4) photolyse

imine analogue of the (6-4) photoproduct (T(6-4)TNH2), in which the carbonyl group is replaced with an iminium cation, is not repaired by the (6-4) photolyase, even in the presence of a 10fold molar excess of the enzyme, although the enzyme binds to the oligonucleotide with considerable affinity. Carbonyl group of the 3' pyrimidone ring plays an important role in the (6-4) photolyase reaction

(6-4) photolyases repair (6-4) pyrimidine-pyrimidone photoproducts. CmPHR1 exhibits a repair activity of both (6-4)-ssDNA and (6-4)-dsDNA. Cryptochromes CmPHR2 and CmPHR5 show CPD repair activity only on ssDNA, and have no repair activity when CPD-damaged dsDNA is used as a substrate

(6-4) photolyases repair (6-4) pyrimidine-pyrimidone photoproducts. CmPHR1 exhibits a repair activity of both (6-4)-ssDNA and (6-4)-dsDNA. Cryptochromes CmPHR2 and CmPHR5 show CPD repair activity only on ssDNA, and have no repair activity when CPD-damaged dsDNA is used as a substrate

cyclobutane pyrimidine dimer-photolyase (EC 4.1.99.3) or 6-4PP-photolyase are able to prevent UV-induced apoptosis in cells deficient for nucleotide excision repair to a similar extent, while in nucleotide excision repair-proficient cells UV-induced apoptosis is prevented only by cyclobutane pyrimidine dimer-photolyase, with no effects observed when pyrimidine-(6-4)-pyrimidone photoproducts are removed by the specific photolyase. Both cyclobutane pyrimidine dimers and pyrimidine-(6-4)-pyrimidone photoproducts contribute to UV-induced apoptosis in nucleotide excision repair-deficient cells, while in nucleotide excision repair-proficient cells, cyclobutane pyrimidine dimers are the only lesions responsible for UV-killing, probably due to the rapid repair of pyrimidine-(6-4)-pyrimidone photoproducts by nucleotide excision repair

Resonance Raman spectra of (6-4) photolyase having neutral semiquinoid and oxidized forms of FAD. Density functional theory (DFT) calculations are carried out on the neutral semiquinone. The marker band of a neutral semiquinone at 1606 cm-1 in H2O, splits into two comparable bands at 1594 and 1608 cm-1 in D2O, and similarly, that at 1522 cm-1 in H2O does into three bands at 1456, 1508, and 1536 cm-1 in D2O. This D2O effect is recognized only after being oxidized once and photoreduced to form a semiquinone again, but not by simple H/D exchange of solvent. Some Raman bands of the oxidized form are observed at significantly low frequencies (1621, 1576 cm-1) and with band splittings (1508/1493, 1346/1320 cm-1). These Raman spectral characteristics indicate strong H-bonding interactions (at N5-H, N1), a fairly hydrophobic environment, and an electron-lacking feature in benzene ring of the FAD cofactor, which seems to specifically control the reactivity of (6-4) photolyase

non-covalently bound to the enzyme. Flavin to apoprotein molecular ratio of 64%. FAD is present in three different redox states: the fully oxidized form (FADox, 82%), the neutral semiquinone (14%) and the fully reduced anion (4%)

for activation of Xenopus (6-4) PHR, FADox is first converted to a neutral radical form by light-induced one-electron and one-proton transfers and then into a fully reduced form by light-induced one electron transfer, mechanism, overview

enzyme is also active under high salinity; NH4Cl or NaH2PO4 increase activity of the enzyme. CH3COONa or Na2CO3 strongly decrease the enzyme’s activity. Maximum activity occurs in the presence of NaH2PO4, which is increased eight times than that in the presence of Na2CO3. When the ions that possess stronger ability to donate a proton are added to enzyme reaction buffer, the rate of photoreactivation increases

for activation of Xenopus (6-4) PHR, FADox is first converted to a neutral radical form by light-induced one-electron and one-proton transfers and then into a fully reduced form by light-induced one electron transfer, four different redox states for the FAD chromophore of PHR, mechanism, overview

(6-4) photolyase binds to T[6-4]T in double stranded DNA with high affinity (KD= 10*exp-9) and to T[6-4]T in single-stranded DNA with slightly lower-affinity (KD= 2*10 exp-8). Majority of the T[6–4]T-(6-4) photolyase complex dissociates very slowly (koff= 2.9* 10exp-5/sec). Its absolute action spectrum without a second chromophore in the 350-600 nm region closely matches the absorption spectrum of the enzyme

in this pH range wild-type, K281G mutant and K281R mutant show different activities: The activity of K281G mutant declines sharply and less than 30% of the activity is retained at pH 11.0, whereas K281R mutant and the wild-type shows more tolerance to the high pH (9.0-11.0), and at pH 11.0, 78.5% and 62.3% of the activity are retained, respectively

T(6-4)C lesion containing DNA duplex in complex with the (6-4) photolyase, by the hanging-drop vapour diffusion method, at 18°C, to 2.95 A resolution. Lesion is flipped out of the opened DNA duplex into the active site of the enzyme

two crystal structures of the (6-4) photolyase bound to lesion containing DNA before and after repair, repair does not involve oxetane formation before light-induced electron transfer. The histidine 369, supposed to activate the acylimine, is in a position that does not allow efficient proton donation and hence activation of this substructure

(6-4) photoproduct DNA photolyase activity is detected in Crotalus atrox fibroblast. Activity is considerably enhanced when a UV-damaged DNA affinity column is used for purification. However, the activity is unstable and it is lost during purification or upon storage at -20° or -70°C for 2-3 months

by using a glutathione sepharose column and a Hi Trap Q column. Concentrated fusion protein is cleaved with thrombin from bovine plasma by incubation overnight at 4°C. For chromophore determination, the eluate from the glutathione sepharose column is purified through a Q sepharose column, omitting gel filtration procedure, and concentrated by ultrafiltration

fusion protein is applied to amylose column. At this point the protein is above 90% pure. Further purification can be obtained by applying the eluted material to a 10 ml heparin-agarose column. Maltose-binding protein is removed by treatment with factor Xa protease

due to insolubility problems (6-4) photolyases is overexpressed as a fusion protein in Escherichia coli. Plasmid pXZ1997, a derivative of pMal-c2 containing the Drosophila melanogaster phr(6-4) cDNA fused in frame to the malE gene encoding maltose-binding protein (MBP), is propagated in Escherichia coli strain UNC523 (phr::kan uvrA::Tn10) selecting for ampicillin resistance. Cells are cultured in 2 liter of LB to A600: 0.6–0.8. IPTG is added to 0.3 mM, and incubation continued for 6 h prior to harvesting the cells by centrifugation.

compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant

compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant

compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant

compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant

compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant

compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant

a single T(6-4)T photoproduct in a 10-mer oligonucleotide is photoreactivated by this mutant. Mutant shows similar capacity of photoreactivation compared to wild-type. Over the pH range 6-9 no difference to wild-type. Over the pH range 9-11 the activity of K281G mutant declines sharply and less than 30% of the activity is retained at pH 11.0

a single T(6-4)T photoproduct in a 10-mer oligonucleotide is photoreactivated by this mutant. Mutant shows similar capacity of photoreactivation compared to wild-type. Over the pH range 6-9 no difference to wild-type. Over the pH range 9-11 the K281R mutant and the wild-type show more tolerance to the high pH (9.0-11.0), and at pH 11.0, 78.5% and 62.3% of the activity are retained, respectively

light-dependent repair of UV-induced (6-4) photoproducts is investigated in an excision repair-deficient Arabidopsis mutant. It is demonstrated that (6-4) photoproducts are efficiently eliminated in a light-dependent manner which occurs in the presence of blue light (435 nm) but not upon exposure to light of longer wavelengths

construction of a theoretical 3D model shows that the protein has a FAD-binding domain. Although the amino acids identity between Dunaliella salina (6-4)photolyase and Escherichia coli CPD photolyase is just 23%, the backbone structure shows a high similarity in overall folding, suggesting a parallel photoreversal mechanism in the two enzymes

expression of H64PRH does not show any photoreactivating effects on the survival of UV-irradiated Escherichia coli. Using a gel shift assay with with un-irradiated and UV-irradiated DNA probes it is shown that H64PRH protein does not possess any binding activity to either DNA probe

sequencing of the cDNA clone reveals an open reading frame of encoding a protein of 526 amino acids (60600 Da) cDNA shows 58-54% amino acid identity to Drosophila (6-4)photolyase and its human homologue and 20-24% identity to the class I CPD photolyase